1. Field
The present invention relates to a detection method of glycoproteins that exhibit an alteration in glycosylation in the course of tumorigenesis and cancer metastasis. More specifically, the present invention relates to a mass spectrometry (MS)-based identification of N-linked glycoproteins that are modified during cancer development and progression.
2. Discussion of the Related Technology
Two-dimensional electrophoresis allowed to identify and characterize proteins with ease and in a high-throughput manner when connected to such highly sophisticated analyzers as MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry and amino acid sequencers, whose developments have facilitated the ‘post-genomics proteomics era’. However, the proteomics approaches have a limitation in that they present a fixed state of proteome rather than revealing a biological dynamism. In fact, signal transduction pathways in cells generally exhibits an dynamism in the expression level of proteins and post-translational modification (PTM).
Moreover, it is often difficult to monitor the proteins on the signal transduction pathway by displaying 2-D gels since the proteins exist sparsely in cells. Protein glycosylation allows to overcome these limitations and to observe cells on a dynamism-basis. Changes in proteins level that can not be discerned by simple staining are easily monitored when alterations in glycosylation are chased with a specific lectin.
Recently, this approach, termed ‘glycomics’, has emerged as a promising field that help overcome the limitations of the conventional proteomics. Glycomics is mainly based on pursuit of alterations in protein glycosylation, a major class of post-translational modification.
One of the biological disturbances is an aberrant glycosylation of proteins, which is induced by a signal of a certain oncogene and causes, in turn, a dysfunction of cell adhesion, cell-cell recognition and eventually tumorigenesis and cancer malignancy (Hakomori and Kannagi, 1983, J. Natl. Cancer Inst., 71:231-251; Feizi, 1985, Nature, 314:53-57). Matured mRNA is translated on an endoplasmic reticulum, where core glycan moiety of N-linked glycosylation is also generated. After that, the glycoproteins are translocated to Golgi body where supplementary glycans are attached by such glycosyltransferase as illustrated in FIG. 1. The glycosyltransferases are activated by a signal elicited by the specific oncogenes, such as ras, raf, ets, and so on. One of the glycosyltransferases that draw interests is FUT8, which catalyzes an addition of fucose to the core N-acetylglucosamine (GlcNAc) of N-glycan. Alpha-fetoprotein (AFP) and antitrypsin are widely known to exemplify the importance of a fucosylation in cancer malignancy (Miyoshi, E., Ko, J. H. et al., 1999, Biochim. Biophys. Acta 1473: 9-20). FUT8 (Fucosyl transferase 8) responsible for the fucosylation was purified and the cDNA was cloned by Taniguchi group and when FUT8-transfected Hep3B (Hep3B-FT) was injected into the spleen of athymic mice, tumor formation in the liver was dramatically suppressed in the transfectants compared to the parental cells (Miyoshi, E., Ko, J. H. et al., 1999, Cancer Research 59: 2237-2243). Lens culinaris agglutinin (LCA) bind the fucose attached to the innermost GlcNAc of N-glycan and also core N-glycan like Con A.
The aberrant glycosylation induced by N-acetylglucosaminyltransferase V (GnT-V) is a representative example of such protein modifications as are implicated in the tumor invasion and metastasis (Dennis et al., 1987, Science, 236:582-585). Following the attachment of core glycans on endoplasmic reticulum, the supplementary glycans are decorated in Golgi body primarily by 6 types of N-acetylglucosaminyltranferase (I-VI). Of these, GnT-V is known to be associated with metastatic potential of cancer cells by modifying the target glycoproteins and changing the properties of recognition and cell adhesion of cancer cells.
GnT-V was first noticed by the report of Dennis et al. (1987) that the β1,6 branches were remarkably represented as cancer tissues were growing or during metastasis (Dennis, et al., 1987, Science, 236: 582-585). A cell surface protein gp130 is one or major target proteins of GnT-V and shows high metastasis activity when β1,6 N-acetylglucosamine is added. GnT-V knockout mice were established in which GnT-V was deficient in their embryonic stem (ES) cells and to which polyomavirus middle T antigen (reffered “PyMT” hereinafter) viral oncogene was introduced in order to induce a cancer. Resultingly, the growth of cancer and metastasis induced by PyMT were remarkably inhibited in GnT-V knockout mice comparing with another normal mice group in which only PyMT was over-expressed (Granovsky et al., 2000, Nature Med., 6: 306-312), and the growth of β1,6 branch caused high metastasis especially in mice with breast cancer. As shown above, GnT-V relates to cancer metastasis and represents high matastasis activity in various tissues. GnT-V was purified in the human lung cancer cell and the mouse kidney, the cDNA was cloned, and the promoter and genomic structure was resolved (Gu et al., 1993, J Biochem, 113:614-619; Soreibah et al., 1993, J. Biol. Chem., 268:15381-15385; Kang et al., 1996, J. Biol. Chem., 271:26706-26712). The inventor of the present invention has reported that the transcription factor ets-1 is involved in the expression of GnT-V (Ko, et al., 1999, J. Biol. Chem., 274(33): 22941-22948). Limitations of the drug-based cancer treatment triggered a paradigm shift in the medical cancer researches toward early detection at a curable stage, and the aberrant glycoproteins are being tested for use as a cancer early-detection marker.
In accordance with this, the inventors of the present invention have reported a list of candidate proteins from cancer cells with high GnT-V overexpression that show an attachment of β1,6-GlcNAc by GnT-V using 2-dimensional electrophoresis and mass spectrometry (Kim et al., 2004, Proteomics, 4: 3353-3358; Kim et al., 2006, Proteomics, 6: 1187-1191). However, the previous gel-based resolution procedures innately have a high incidence of false-positive rates in the identification of sparse proteins. To overcome this, the inventors completed the present invention by enriching β1,6-GlcNAc-carrying glycoproteins using L-PHA lectin to identify them.